The biosynthesis of vitamin K-dependent proteins includes several posttranslational processing steps before a mature functional protein is obtained.
Vitamin K is a necessary cofactor for the gamma-carboxylation of glutamic acid residues in these vitamin K-dependent proteins, including the procoagulant factors thrombin, factor VII, IX, and X; the anticoagulants protein C and protein S; and other proteins such as osteocalcin (bone Gla protein), matrix Gla protein, and proline-rich Gla protein 1. This carboxylation is required for normal hemostasis, because it enables calcium binding and attachment of the procoagulants and anticoagulants to phospholipids.
Gamma-glutamyl carboxylase is an integral membrane microsomal enzyme located in the rough endoplasmic reticulum. It carboxylates glutamate residues located in the Gla domain of the vitamin K-dependent proteins. Human gamma-glutamyl carboxylase cDNA has recently been isolated and sequenced (Wu S M et al. Science 254:1634, 1991). Studies of the biosynthesis of Vitamin K-dependent proteins in BHK and CHO cells, show that the carboxylase is present in both the endoplasmatic reticulum (ER) and the Golgi complex, and that the propeptide, containing the carboxylase recognition site is cleaved after completion of the gamma-carboxylation.
It has been speculated whether the propeptide can stimulate the carboxylase activity (Sigiura, I. et al. (1997) Proc. Natl. Acad. Sci., 9, 9069-9074, Knobloch and Suttie (1987) J. Biol. Chem. 262, 15334-15337, Furie et al (1999) Blood, 93, 1798-1808).
Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation “cascade” are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as “active factors,” and are designated by the addition of a lower case “a” suffix (e.g., activated factor VII (FVIIa)).
Activated factor X (FXa) is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways, that promote the activation of FX. The “intrinsic pathway” refers to those reactions that lead to thrombin formation through utilization of factors present only in plasma. A series of protease-mediated activations ultimately generates factor IXa which, in conjunction with factor VIIIa, cleaves FX into FXa. A similar proteolysis is effected by FVIIa and its co-factor, tissue factor, in the “extrinsic pathway” of blood coagulation. Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it can complex with FVIIa to catalyze FX activation or factor IX activation in the presence of Ca++ and phospholipid. While the relative importance of the two coagulation pathways in hemostasis is unclear, in recent years FVII and tissue factor have been found to play a pivotal role in the regulation of blood coagulation.
FVII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is clot inactive. Single-chain FVII may be converted to two-chain FVIIa by FXa, factor XIIa, factor IXa or thrombin in vitro. FXa is believed to be the major physiological activator of FVII. Like several other plasma proteins involved in hemostasis, FVII is dependent on vitamin K for its biosynthesis, which is required for the gamma-carboxylation of 10 glutamic acid residues in the amino terminus of the protein. The intracellular post-translational processing of FVII takes place in the endoplasmatic reticulum (ER) and the Golgi complex. Besides the vitamin K-dependent gamma-carboxylation, FVII is subjected to limited proteolysis to remove the N-terminal propeptide, and glycosylation of asparagine-145 and -322, and serine-52 and -60 (FIG. 1).
The gamma-carboxylated glutamic acid (Gla) residues are required for the metal-associated interaction of FVII with phospholipids.
In the presence of tissue factor, phospholipids and calcium ions, the two-chain FVIIa rapidly activates FX or factor IX by limited proteolysis.
Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating factors Va and VIIIa in the coagulation cascade. Human protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. This single chain precursor molecule undergoes multiple post-translational modifications including carboxylation of nine glutamic acid residues, resulting in nine Gla residues.
Protein S also exhibits anticoagulant activity in in vitro clotting assays. Protein S demonstrates anticoagulant cofactor activity for activated protein C. Protein S has also been shown to be an anticoagulant factor in the absence of activated protein C as it can inhibit prothrombinase activity in assays free of activated protein C and binds to Factor Va or Factor Xa and functions as an anticoagulant without activated protein C. Protein S is physiologically a very important antithrombotic factor since hereditary or acquired deficiencies of protein S are associated with venous and arterial thrombotic disease. A deficiency of free protein S with a normal level of total protein S has been described in some patients with thrombotic disease.
It is often necessary to selectively block the coagulation cascade in a patient. Anti-coagulants such as protein C or protein S may be used, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), a patient at risk for acute thrombosis, protein S deficiency, sepsis, inflammation, cancer, patients undergoing surgery and a host of other medical disorders.
Osteocalcin is composed of 49 amino acid residues which include three Gla residues. The function of this protein is thought to be to suppress excessive mineralization. Osteocalcin is a bone-specific protein that is secreted by osteoblasts. A fraction of newly synthesized osteocalcin is released into the bloodstream, where its concentration correlates with the indices of osteoblastic activity and bone formation rate. In humans, changes in circulating osteocalcin levels have been associated with metabolic bone diseases such as osteoporosis and hyperparathyroidism.
Matrix Gla Protein (MGP) is composed of 79 amino acids including 5 Gla residues. This protein is usually found in demineralized matrix and believed to have a certain function in the initiation of bone formation.
There is still a need in the art for improved systems for the production of recombinant vitamin K-dependent proteins and particular recombinant coagulation factors. The present invention fulfills this need by providing a method that gives a more efficient, faster production and/or higher yield of recombinant vitamin K-dependent proteins, in particular FVII.